Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/20—Aspartic acid; Asparagine

Definitions

• L-aspartic acid is an essential starting material for the synthesis of the dipeptide Aspartame, an artificial sweetener with a +/- 200-fold sweetening power of Sucrose.
• a variety of chemical have been used for the synthesis of aspartame and enzymatic processes, in which L-aspartic acid in particular and phenylalanine methyl ester in a variety of ways, such as with or without enzymatic catalysis.
• L-aspartic acid in particular and phenylalanine methyl ester
• Z-Cl benzyloxycarbonyl chloride
• Aspartic acid is how described for example in EP-A-0 127 940, from maleic acid, to fumaric acid isomerized, enzymatically via the ammonium L-aspartate and subsequent crystallization in the presence of an acid.
• the object of the present invention was therefore to find a method for which avoided the ammonium ion-containing wastewater, the number of Steps to Z-L aspartic acid disodium salt reduced and the required Specifications are met.
• this task could be solved by a process in which, starting from fumaric acid, ammonium L-aspartate directly into the Disodium salt of L-aspartic acid with simultaneous quantitative recycling of ammonia is transferred.
• fumaric acid is in a enzymatic reaction converted to ammonium L-aspartate.
• the starting product for the process according to the invention fumaric acid can be produced, for example, by isomerization of maleic acid.
• fumaric acid is stirred into an inert diluent up to the solubility limit, so that a suspension is obtained.
• Suitable inert diluents are water, water / ethanol or water / acetone mixtures and the like. Water is preferably used.
• Ammonia is then liquefied into the fumaric acid suspension at room temperature or introduced in the form of a 10 to 35% by weight solution, as a result of which the temperature rises to 60 ° C. and a pH value between 8 and 9 is established.
• An aqueous 20 to 30% by weight ammonia solution is preferably used. Ammonia is added in excess, preferably in a 1-30% excess.
• the enzyme aspartase or an aspartase-producing microorganism is then stirred into the system thus obtained, preferably a solution, at 20 to 60 ° C., preferably at 30 to 50 ° C.
• a solution is obtained by the addition of ammonia, since in the case of a suspension due to adsorption of the enzyme and consequent loss of activity, more enzyme is required.
• 30 to 50 IU enzyme activity per mole of fumaric acid are required.
• Aspartase-producing microorganisms are, for example, Pseudomonas fluorescens, Protens vulgaris, Pseudomonas aeruginosa, Serratia marcescens, Bacterium succinium, Bacillus subtilis, Aerobacter aerogenes, Micrococcus sp. Escherichia coli and others Further suitable aspartase-producing microorganisms are described, for example, in US Pat. No. 3,791,926 and US Pat. No. 3,198,712. Purified or synthetic aspartase can also be used in the method according to the invention.
• the enzyme or the aspartase-producing microorganism can be added in liquid or in immobilized form, as described, for example, in EP 0 127 940.
• the end of the reaction can be determined, for example, photometrically, the ammonium L-aspartate-containing solution is optionally cooled to 20 to 30 ° C. and sodium hydroxide is added in step 2 (b).
• the amount of sodium hydroxide added is dependent on the desired sodium salt or on the desired pH of the product solution. Both the monosodium and disodium salt and mixtures thereof are suitable for the further reaction.
• Sodium hydroxide can be used in solid form or as sodium hydroxide solution. Sodium hydroxide solution is preferably used.
• the reaction solution heats up by about 10 - 30 ° C.
• ammonia from the reaction mixture is preferred continuously using a thin film evaporator, falling film evaporator or a short path evaporator removed by distillation.
• the reaction or reflux temperature is 90-120 ° C, preferably 100-110 ° C.
• Step c According to this procedure when separating the ammonia is a practically quantitative removal of the ammonia (residual content ⁇ 100 ppm) guaranteed and racemization reactions (D-Asp ⁇ 0.1% by weight) and one The yellowing of the remaining solution and cleavage to fumaric acid are suppressed or practically completely prevented.
• the sodium L-aspartate solution thus obtained is then in the next step d) mixed with benzyloxycarbonyl chloride (Z-Cl) at a temperature of 30 to 60 ° C, whereby a pH value between 9 and 14 is preferably kept between 11 and 12.
• Z-Cl can do it be added in an equimolar amount, but also in a slight excess.
• the reaction mixture is still 0.5 to 4 hours the reaction temperature left and then to room temperature cooled, whereupon phase separation occurs.
• the aqueous phase is then washed, for example with methyl tert-butyl ether, diisobutyl ether, toluene or other water-immiscible solvents.
• the organic phase is discarded and ZL-aspartic acid is obtained as the disodium salt in aqueous solution.
• a vacuum is applied at room temperature to remove any residual amounts of organic solvents in the aqueous phase.
• the process according to the invention gives ZL-aspartic acid disodium salt as a clear, slightly yellowish solution which, if required, can be used for further processing to aspartame by coupling with phenylalanine. Waste water containing ammonium ions is avoided by the process according to the invention and ammonia is quantitatively recycled for further enzymatic conversions of fumaric acid.
• the disodium salt solution of Z-L-aspartic acid thus obtained corresponds to all Specifications regarding the residual content of fumaric acid ( ⁇ 0.3% by weight) and ammonia ( ⁇ 100 ppm), color (color number APHA ⁇ 25) and optical purity (D-Asp. ⁇ 0.1% by weight).
• time ml in template Bottom temperature (° C) ml head ml swamp 0 h 1000 88.2 after 40 min 500 94.3 230 270 1 h 23 min 0 82.3 480 510
• the distilled ammonia was introduced into a fumaric acid suspension, which served as a starting solution for a further reaction. The residual ammonia content was 20 ppm. The content of fumaric acid was ⁇ 0.04% by weight, malic acid and asparagine could not be detected.
• the aqueous solution was treated twice with 92 ml each time of methyl tert-butyl ether. The organic phase was discarded; the aqueous phase was freed from solvent residues at 20 ° C. and 20 mbar in a Rotavapor for 30 minutes. 101 g of a slightly yellowish, clear solution were obtained. The content was 30.8% by weight of disodium Z (L) aspartate.
• Example 1 b) and c) Analogously to Example 1 b) and c) was in 1300 ml of a solution obtained in 1a) after cooling to 25 ° C, 588 g of 20% by weight sodium hydroxide solution within Introduced 15 minutes. The solution warmed to 50 °. At The total amount of ammonia was distilled by atmospheric pressure analogously to Example 1c removed and the reaction solution concentrated to 1200 ml. Residual ammonia content ⁇ 100 ppm.
• a monosodium L-aspartate solution containing 25.9% by weight of L-aspartic acid was obtained. No racemization was observed. The fumaric acid content was ⁇ 0.1% by weight, malic acid and asparagine could not be detected.

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• 1998
  • 1998-02-02 AT AT0016398A patent/ATA16398A/de not_active Application Discontinuation
• 1999
  • 1999-01-13 CZ CZ99105A patent/CZ10599A3/cs unknown
  • 1999-01-15 SK SK54-99A patent/SK5499A3/sk unknown
  • 1999-01-28 EP EP99101849A patent/EP0945517A3/fr not_active Withdrawn
  • 1999-02-01 HU HU9900215A patent/HUP9900215A2/hu unknown

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